The invention relates to molecular biology and in particular to genes and proteins involved in vertebrate neural development and to methods for classifying and prognosticating neuroectodermal tumors.
Transcription factors of the basic-helix-loop-helix (bHLH) family are implicated in the regulation of differentiation in a wide variety of cell types, including trophoblast cells (Cross et al., Development 121:2513-2523, 1995), pigment cells (Steingrimsson et al., Nature Gen. 8:251-255, 1994), B-cells (Shen, C. P. and T. Kadesch., Molec. and Cell. Biol. 15:3813-3822, 1995; Zhuang et al., Cell 79:875-884, 1994), chondrocytes and osteoblasts (Cserjesi et al., Development 121:1099-1110, 1995; Tamura, M. and M. Noda., J. Cell Biol. 126:773-782, 1994), and cardiac muscle (Burgess et al., Develop. Biol. 168:296-306, 1995; Hollenberg et al., Molec. and Cell. Biol. 15:3813-3822, 1995). bHLH proteins form homodimeric and heterodimeric complexes that bind with DNA in the 5xe2x80x2 regulatory regions of genes controlling expression.
Perhaps the most extensively studied sub-families of bHLH proteins are those that regulate myogenesis and neurogenesis. The myogenic bHLH factors, (MyoD, myogenin, Myf5, and MRF4), appear to have unique as well as redundant functions during myogenesis (Weintraub, H., Cell 75:1241-1244, 1993; Weintraub et al., Science 251:761-766, 1991). It is thought that either Myf5 or MyoD is necessary to determine myogenic fate, whereas myogenin is necessary for events involved in terminal differentiation (Hasty et al., Nature 364:501-506, 1993; Nabeshima et al., Nature 364:532-535, 1993; Rudnicki et al., Cell 75:1351-1359, 1993; Venuti et al., J. Cell Biol. 128:563-576, 1995). Moreover, Myf expression has been observed in a number of rhabdomyosarcomas, and has been proposed as a marker for that category of tumor (Clark et al., Br. J. Cancer 64:1039-1042, 1991).
Recent work on neurogenic bHLH proteins suggests parallels between the myogenic and neurogenic sub-families of bHLH proteins. Genes of the Drosophila melanogaster achaete-scute complex and the atonal gene have been shown to be involved in neural cell fate determination (Anderson, D. J., Cur. Biol. 5:1235-1238, 1995; Campuzano, S. and J. Modolell., Trends in Genetics 8:202-208, 1992; Jaman et al., Cell 73:1307-1321, 1993), and the mammalian homologs, MASH1 and MATH1, are expressed in the neural tube at the time of neurogenesis (Akazawa et al., J. Biol. Chem. 270:8730-8738, 1995; Lo et al., Genes and Dev. 5:1524-1537, 1991). Two related vertebrate bHLH proteins, neuroD1 and NEX-1/MATH-2, are expressed slightly later in CNS development, predominantly in the marginal layer of the neural tube and persisting in the mature nervous system (Bartholoma, A. and K. A. Nave, Mech. Dev. 48:217-228, 1994; Lee et al., Science 268:836-844, 1995; Shimizu et al., Eur. J. Biochem. 229:239-248, 1995). NeuroD1 was also cloned as a factor that regulates insulin transcription in pancreatic beta cells and named xe2x80x9cBeta2xe2x80x9d (Naya et al., Genes and Dev. 9:1009-1019, 1995). Constitutive expression of neuroD1 in developing Xenopus embryos produces ectopic neurogenesis in the ectodermal cells, indicating that neuroD genes are capable of regulating a neurogenic program. A neuroD1 homolog having 36,873 nucleotides has been identified in C. elegans (Lee et al., 1995; Genbank Accession No. 010402), suggesting that this molecular mechanism of regulating neurogenesis may be conserved between vertebrates and invertebrates.
A human achaete-scute homolog has been identified and cloned whose predicted protein of 238 amino acids is 95% homologous to the mouse hash1 gene (Ball et al., Proc. Natl. Acad. Sci. USA 90:5648-5652, 1993). Northern blots revealed that transcripts from this bHLH gene were detectable in two types of cancer with neuroendocrine features, namely small cell lung cancer, and the calcitonin-secreting medullary thyroid carcinoma (id.). Thus, hash1 was proposed to provide a marker for cancers with neuroendocrine features.
Primitive neuroectodermal tumors (PNET) are the most common of the malignant central nervous system tumors that occur in children (Biegel et al., Genes, Chrom. and Cancer 14: 85-96, 1995). Both supratentorial and infratentorial PNETs occur. Medulioblastoma, an infratentorial tumor that expresses neuronal intermediate filaments, synaptic vesicle proteins, growth factor receptors, and adhesion molecules, is the prototypic PNET. The location (posterior fossa) and properties of medulloblastomas suggests that they arise from neuroblasts that escape terminal differentiation (Trojanowski, J. Q., et al. Mol. Chem. Neuropathol. 17:121-135, 1992). Abnormalities in chromosome 17 have been observed in PNET biopsies, though the significance of these abnormalities has not been determined (Biegel et al., 1995; Schultz, et al., Genes, Chrom. and Cancer 16:196-203, 1996). Factors presently used to classify and prognosticate brain tumors include location, histopathology, patient age, and biological behavior of the tumor. However, such bases for tumor identification are not always sufficient for accurate prognostication. Supratentorial and infratentorial PNETs cannot always be distinguished, though they may respond differently to therapy. (Heideman, R. L. et al. xe2x80x9cTumors of the central nervous system.xe2x80x9d In: P. A. Pizzo and D. G. Poplack (eds.), Principles and Practice of Pediatric Oncology, 2nd. ed., pp. 633-682, 1993; Rorke, L. B., et al. Cancer 56:1869-1886, 1996; Packer, R. J., et al. J. Neurosurg. 81:690-698, 1994; Cohen, B. H. et al. J. Clin. Oncol. 13:1687-1696, 1995).
Because different types of brain tumors respond differently to various therapeutic regimens, accurate classification is highly useful to the physician in determining the best course of treatment for a particular patient. Medulloblastomas about half of the time are confined to the bridge that connects the two halves of the cerebellum (vermis). Medulloblastoma often is an aggressive and highly malignant type of tumor, and may invade other portions of the brain and spinal column. Thus, diagnosis based on location is not always reliable. Histologically, medulloblastoma is highly cellular, consisting of undifferentiated small dark round cells. Other PNETs with an identical histologic appearance, occurring predominantly in infants, are found at other locations in the brain. Thus, markers for a more accurate determination of PNET origin would facilitate the assignment of the therapeutic regimen most likely to be effective. Currently, no effective biologic markers exist for facilitating the stratification of treatment groups, assisting in prognosis, or providing targets for therapeutic intervention (Heideman et al., 1993).
The presently disclosed neuroD proteins represent a new sub-family of bHLH proteins and are implicated in vertebrate neuronal, endocrine and gastrointestinal development. Mammalian and amphibian neuroD proteins have been identified, and polynucleotide molecules encoding neuroD proteins have been isolated and sequenced. NeuroD genes encode proteins that are distinctive members of the bHLH family. In addition, the present invention provides a family of neuroD proteins that share a highly conserved HLH region. Representative polynucleotide molecules encoding members of the neuroD family include neuroD1, neuroD2 and neuroD3.
A representative nucleotide sequence encoding murine neuroD1 is shown in SEQ ID NO:1. The HLH coding domain of murine neuroD1 resides between nucleotides 577 and 696 in SEQ ID NO:1. The deduced amino acid sequence of murine neuroD1 is shown in SEQ ID NO:2. There is a highly conserved region following the helix-2 domain from amino acid 150 through amino acid 199 of SEQ ID NO:2 that is not shared by other bHLH proteins.
A representative nucleotide sequence encoding Xenopus neuroD1 is shown in SEQ ID NO:3. The HLH coding domain of Xenopus neuroD1 resides between nucleotides 376 and 495 in SEQ ID NO:3. The deduced amino acid sequence of Xenopus neuroD1 is shown in SEQ ID NO:4. There is a highly conserved region following the helix-2 domain from amino acid 157 through amino acid 199 of SEQ ID NO:4 that is not shared by other bHLH proteins.
Representative nucleotide and deduced amino acid sequences of the human neuroD family are shown in SEQ ID NOS:8-15. Representative nucleotide and deduced amino acid sequences of a human homolog of murine neuroD1 are shown in SEQ ID NOS:8 and 9 (partial genomic sequence) and SEQ ID NOS:14 and 15 (human neuroD1 cDNA). Representative nucleotide and deduced amino acid sequences of the human and murine neuroD2 are shown in SEQ ID NOS: 10 and 11, and 16 and 17, respectively. Representative nucleotide and deduced amino acid sequences for human neuroD3 are shown in SEQ ID NOS:12 and 13. The disclosed human clones, 9F1(and its corresponding cDNA HC2A; now referred to as human neuroD1) and 14B1 (now referred to as human neuroD2), have an identical HLH motif: Amino acid residues 117-156 in SEQ ID NO:9 and 15, and residues 137-176 in SEQ ID NO:11 (corresponding to nucleotides 405-524 of SEQ ID NO:8 and SEQ ID NO:14, and nucleotides 463-582 of SEQ ID NO:10). Comparison of the deduced amino acid sequences of these neuroD genes shows that human neuroD3 contains an HLH domain between amino acid residues 108-147 of SEQ ID NO:13 (corresponding to nucleotides 376-495 of SEQ ID NO:12) and that murine neuroD2 contains an HLH domain between amino acids residues 138-177 of SEQ ID NO:17 (corresponding to nucleotides 641-760 of SEQ ID NO:16). The HLH domain of murine neuroD2 is identical to that of the human neuroD1 and human neuroD2 proteins. Similar analyses indicated that mouse neuroD3 contains an HLH domain between amino acid residues 109-148 of SEQ ID NO:22 (corresponding to nucleotides 425-544 of SEQ NO:21).
Expression of several bHLH genes were analyzed in cerebellar and cerebral primitive neuroectodermal tumors (PNETs), gliomas, and in cell lines derived from a variety of neuroectodermal tumors. Generally, the observed patterns of neuroD expression distinguished subclasses of neuroectodermal tumors and generally recapitulated gene expression patterns of tissues from which neuroectodermal tumors arise. For example, a strildng association of neuroD3 with aggressive cases of medulloblastoma was noted, suggesting that neuroD3 provides a useful prognosticator for aggressive medulloblastoma.
The subject invention provides three representative members of the neuroD family of genes, namely, neuroD1, neuroD2 and neuroD3 (also called xe2x80x9cneurogeninxe2x80x9d). More specifically, the invention provides murine neuroD1 (SEQ ID NOS:1 and 2, Xenopus laevis neuroD1 (SEQ ID NOS:3 and 4, human neuroD1 (SEQ ID NOS:8, 9, 14, and 15, human neuroD2 (SEQ ID NOS:10 and 11), human neuroD3 (SEQ ID NOS:12 and 13), murine neuroD2 (SEQ ID NOS: 16 and 17), and murine neuroD3 (SEQ ID NOS:21 and 22).
Provided are methods of classifying human neuroectodermal tumors by analyzing a sample of the tumor, such as, for example, a biopsy or a sample of an excised tumor. For this analysis, the expression of at least one basic helix loop (bHLH) gene is measured in the sample, and the tumor is classified as belonging to a particular subclass of neuroectodermal tumor in accord with the observed expression pattern. Examples of neuroectodermal tumor subclasses amenable to this analysis include supratentorial PNETs, such as neuroblastoma, as well as infratentorial PNETs, such as medulloblastoma. Predetermined profiles of bHLH expression associated with particular subclasses of neuroectodermal tumors are established by collecting tumor samples that have been classified by conventional methods, analyzing them for bHLH expression, and correlating the observed patterns of expression with the various subclasses of tumor. It is demonstrated that bHLH expression in several instances correlates with tumor subclass, and it is contemplated that these methods will be applicable to additional subclasses of neuroectodermal tumors. NeuroD genes whose measurement is useful in this context include neuroD1, neuroD2, neuroD3, but it is contemplated that additional neuroD family members will also contribute to this method of tumor classification. Hence, at least one, and preferably several, bHLH genes are analyzed in each tumor sample. For example, individual tumor samples may be analyzed for the expression of neuroD1, neuroD2, and neuroD3. It is determined that the tumor belongs to a given subclass of neuroectodermal tumors if the bHLH gene or genes expressed in the sample corresponds to a predetermined profile of basic helix loop helix expression associated with that subclass of neuroectodermal tumor.
Tumor samples typically will be obtained during excision of the tumor from the patient, but may be obtained by other means, such as a biopsy or a spinal tap. Alternatively, the tumor sample may be obtained from the site of a metastatic tumor that originated from a neural tumor but that is located outside the primary site of disease. Such metastatic tumors may appear in any part of the body, but most often are found in the spinal cord. Tumor samples also may be obtained from spinal fluid, which may be analyzed directly, or may be subjected to centrifugation to collect cellular material which in turn is analyzed.
The term xe2x80x9cbHLH expressionxe2x80x9d refers to expression of the bHLH gene in the form of transcripts and/or polypeptide products. bHLH expression thus can be measured by using assays that detect bHLH RNA or polypeptides. bHLH transcripts typically are measured by amplification using polymerase chain reaction (PCR), or hybridization under stringent conditions of RNA from the tumor sample with DNA or RNA probes corresponding specifically to the nucleotide sequences of non-conserved regions of cloned bHLH cDNAs, genes, or subportions thereof By xe2x80x9cnon-conserved regionxe2x80x9d is meant that the probe does not encode the conserved bHLH domain itself. Hybridization methods for detecting bHLH transcripts include hybridization in solution, Northern blot analysis, dot blot or slot blot analysis, in situ hybridization, hybridizations wherein the probe is anchored to a solid substratum, liquid hybridization wherein the formed duplexes are subsequently captured on a solid substratum, or other methods of hybridization. Probes may be labeled directly, e.g., with radioisotopes or biotin, or may contain nucleotide sequences complementary to a secondary probe that itself is labeled.
The term xe2x80x9ccapable of hybridizing under stringent conditionsxe2x80x9d means that the probe anneals under stringent hybridization conditions to the target nucleic acid molecule. Individual probe molecules are generally between 7-1000 nucleotides in length, and preferably are between 10 and 650 nucleotides in length. Probe molecules longer than 1000 nucleotides may be used, but it is preferable that these be sheared to fragments of 200-500 nucleotides in length prior to use. Long DNA molecules to be used as probes may be sheared mechanically, enzymatically, or by brief alkali treatment.
xe2x80x9cStringent hybridizationxe2x80x9d is generally understood in the art to mean that the nucleic acid duplexes that form during the hybridization reaction are perfectly matched or nearly perfectly matched. Several rules governing nucleic acid hybridization have been well established. For example, it is standard practice to achieve stringent hybridization for polynucleotide molecules  greater than 200 nucleotides in length by hybridizing at a temperature 15xc2x0-25xc2x0 C. below the melting temperature (Tm) of the expected duplex, and 5xc2x0-10xc2x0 C. below the Tm for oligonucleotide probes (e.g., Sambrook et al., Molecular Cloning, [2d ed.], Cold Spring Harbor Laboratory Press, 1989, which is hereby incorporated by reference; see Section 11.45). Under such conditions, stable hybrid duplexes will form only if few or no mismatches are present. When Northern or Southern blots are performed, the detection of only well-matched hybrids can be assured by conducting the hybridization step under low or moderate stringency conditions, and then conducting the final wash steps under stringent wash conditions, i.e., at about 10-14xc2x0 C. below the Tm.
The Tm of a nucleic acid duplex can be calculated using a formula based on the % G+C contained in the nucleic acids, and that takes chain length into account, such as the formula Tm=81.5xe2x88x9216.6 (log [Na+])+0.41 (% G+C)xe2x88x92(600/N), where N=chain length (Sambrook et al., 1989, at Section 11.46). It is apparent from this formula that the effects of chain length on Tm is significant only when rather short nucleic acids are hybridized, and also that the length effect is negligible for nucleic acids longer than a few hundred bases.
The term xe2x80x9ccapable of hybridizing under stringent conditionsxe2x80x9d as used herein means that the subject nucleic acid molecules (whether DNA or RNA) anneal under stringent hybridization conditions to an oligonucleotide probe of 15 or more contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:21. Oligonucleotides 15 nucleotides or more in length are extremely unlikely to be represented more than once in a mammalian genome, hence such oligonucleotides can form specific hybrids (see, for example, Sambrook et al., at Section 11.7).
The choice of hybridization conditions will be evident to one skilled in the art and will generally be guided by the purpose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the level of desired relatedness between the sequences. See, for example: Sambrook et al., 1989.; Hames and Higgins, eds., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington D.C., 1985; Berger and Kimmel, eds., Methods in Enzymology, Vol 52, Guide to Molecular Cloning Techniques, Academic Press Inc., New York, N.Y., 1987; and Bothwell, Yancopoulos and Alt, eds., Methods for Cloning and Analysis of Eukcwyotic Genes, Jones and Bartlett Publishers, Boston, Mass. 1990; which are incorporated by reference herein in their entirety. The stability of nucleic acid duplexes is known to decrease with an increased number of mismatched bases, and further to be decreased to a greater or lesser degree depending on the relative positions of mismatches in the hybrid duplexes. Thus, the stringency of hybridization may be used to maximize or minimize the stability of such duplexes. Hybridization stringency can be altered by: adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and/or salt concentration of the wash solutions. For filter hybridizations, the final stringency of hybridization often is determined by the salt concentration and/or temperature used for the post-hybridization washes. In general, the stringency of hybridization reaction itself may be reduced by reducing the percentage of formamide in the hybridization solution. High stringency conditions, for example, may involve high temperature hybridization (e.g., 65-68xc2x0 C. in aqueous solution containing 4-6xc3x97SSC (1xc3x97SSC=0.15 M NaCl, 0.015 M sodium citrate), or 42xc2x0 C. in 50% formamide combined with washes at high temperature (e.g., 5-25xc2x0 C. below the Tm), in a solution having a low salt concentration (e.g., 0.1xc3x97SSC). Low stringency conditions may involve lower hybridization temperatures (e.g., 35-42xc2x0 C. in 20-50% formamide) with washes conducted at an intermediate temperature (e.g., 40-60xc2x0 C.) and in a wash solution having a higher salt concentration (e.g., 2-6xc3x97SSC). Moderate stringency conditions, which may involve hybridization in 0.2-0.3M NaCl at a temperature between 50xc2x0 C. and 65xc2x0 C. and washes in 0.1xc3x97SSC, 0.1% SDS at between 50xc2x0 C. and 55xc2x0 C., may be used in conjunction with the disclosed polynucleotide molecules as probes to identify genomic or cDNA clones encoding members of the neuroD family.
To measure the amount of bHLH protein in a sample, antibody-based methods can be used. For example, either monoclonal or polyclonal antibody directed against the target protein can be used in Western blots, radioimmunoassays (RIA). enzyme-linked immunosorbent assays (ELISA), or the like.
For purposes of the subject invention, bHLH expression is xe2x80x9cdetectedxe2x80x9d if the level of bHLH transcripts or protein is elevated above the background level in the assay that is conducted to measure the transcripts or protein. xe2x80x9cBackground levelxe2x80x9d is the level of signal observed in control reactions in which the target transcript or protein is not present.
In one embodiment of the invention, a method is provided wherein a human neuroectodermal tumor is classified as a medulloblastoma by measuring neuroD1 and neuroD3 expression in a sample of the tumor, and determining that the tumor is a medulloblastoma if both neuroD1 and neuroD3 expression are detected in the sample.
High level expression of neuroD in neuroendocrine tumors and in rapidly proliferating regions of embryonic neural development (see below) indicates that measuring the levels of expression of neuroD or other bHLH genes may provide prognostic markers for assessing the growth rate and invasiveness of a neural tumor. Provided here is a method for prognosticating a human medulloblastoma based on measuring the expression of neuroD3, which is expressed in some but not all medulloblastomas (see Table 1, Example 17). It is determined that the tumor is an aggressive medulloblastoma if neuroD3 expression above the background level is detected in the sample. By xe2x80x9cprognosticatingxe2x80x9d is meant the foretelling of the probable course of a tumor in advance of treatment, and predicting the likelihood that treatment will be successful.
The instant invention may also be used in the identification of sites of metastases. The methods of the invention may be used, for example, to identify cells located outside the primary site (e.g., located in a lymph node or in the bone marrow) that are expressing a constellation of neuroD family members consistent with a tumor from which they are suspected to have originated. The identification of such cells would indicate the presence of metastatic disease. The methods of the invention may also be used similarly to assay for microscopic disease in bone marrow or peripheral blood stem cells harvested for autologous marrow trasplant or stem cell re-infusion. In addition, the methods may be used to determine the degree of tumor cell reduction in marrow or stem cells that have undergone purging to eliminate tumor cells or that have undergone physical selection away from tumor cells.
The neuroD family of genes function during the development of the nervous system. Like MATH1 (Lo et al., Genes and Dev. 5:1524-1537, 1991), the expression of neuroD3 peaks during embryonic development and is not detected in the mature nervous system. NeuroD2 shows a high degree of sequence similarity to both neuroD1 and NEX-1/MATH2, and is similarly expressed both during embryogenesis and in the mature nervous system, demonstrating an expression pattern that partially overlaps with neuroD1. Like neuroD1, neuroD2 when expressed by transfection in Xenopus embryos induces neurogenesis in ectodermal cells. Heterologous expression of neuroD1 and neuroD2 indicates that these highly similar transcription factors demonstrate some target specificity, with the GAP-43 promoter being activated by neuroD2 and not by neuroD1. The partially overlapping expression pattern and target specificity of neuroD1 and neuroD2 suggests that this group of neurogenic transcription factors may contribute to the establishment of neuronal identity in the nervous system by acting on an overlapping but non-congruent set of target genes.
NeuroD proteins are transiently expressed in differentiating neurons during embryogenesis. NeuroD proteins are also detected in adult brain, in the granule layer of the hippocampus and the cerebellum. In addition, murine neuroD1 expression has been detected in the pancreas and gastrointestinal tissues of developing embryos and post-natal mice (see, e.g., Example 14). NeuroD proteins contain the basic helix-loop-helix (bHLH) domain structure that has been implicated in the binding of bHLH proteins to upstream recognition sequences and activation of downstream target genes. Based on homology with other bHLH proteins, the bHLH domain for murine neuroD1 is predicted to reside between amino acids 102 and 155 of SEQ ID NO:2, and between amino acids 101 and 157 of SEQ ID NO:4 for the amphibian neuroD1.
NeuroD proteins are transcriptional activators that control transcription of downstream target genes including genes that among other activities cause neuronal progenitors to differentiate into mature neurons. In the neural stage of the mouse embryo (e10), murine neuroD1 is highly expressed in the neurogenic derivatives of neural crest cells, the cranial and dorsal root ganglia, and postmitotic cells in the central nervous system (CNS). During mouse development, neuroD1 is expressed transiently and concomitant with neuronal differentiation in differentiating neurons in sensory organs such as in nasal epithelium and retina. In Xenopus embryos, ectopic expression of neuroD1 in non-neuronal cells induced formation of neurons. As discussed in more detail below, neuroD proteins are expressed in differentiating neurons and are capable of causing the conversion of non-neuronal cells into neurons. The present invention encompasses variants of neuroD genes that, for example, are modified in a manner that results in a neuroD protein capable of binding to its recognition site, but unable to activate downstream genes. The present invention also encompasses fragments of neuroD proteins that, for example, are capable of binding the natural neuroD partner, but that are incapable of activating downstream genes. NeuroD proteins encompass proteins retrieved from naturally occurring materials and closely related, functionally similar proteins retrieved by antisera specific to neuroD proteins, and recombinantly expressed proteins encoded by genetic materials (DNA, RNA, cDNA) retrieved on the basis of their similarity to the unique regions in the neuroD family of genes.
The present invention provides representative isolated and purified polynucleotide molecules encoding proteins of the neuroD family. Polynucleotide molecules encoding neuroD include those sequences resulting in minor genetic polymorphisms, differences between species, and those that contain amino acid substitutions, additions, and/or deletions. According to the present invention, polynucleotide molecules encoding neuroD proteins encompass those molecules that encode neuroD proteins or peptides that share identity with the sequences shown in SEQ ID NOS:2, 4, 9, 11, 13, 15, and 17.
In some instances, one may employ such changes in the sequence of a recombinant neuroD polynucleotide molecule to substantially decrease or even increase the biological activity of neuroD protein relative to the wild-type neuroD activity, depending on the intended use of the preparation. Such changes may also be directed towards endogenous neuroD polynucleotide sequences using, for example, gene therapy methods to alter the gene product. Such changes are envisioned with regard to neuroD1, neuroD2, neuroD3, or other members of the neuroD gene family.
The neuroD1 proteins of the present invention are capable of inducing the expression in a frog embryo of neuron-specific genes, such as N-CAM , xcex2-tubulin, and Xen-1, neurofilament M (NF-M), Xen-2, tanabin-1, shaker-1, and frog HSCL. As described below in Example 10, neuroD1 activity may be detected when neuroD is ectopically expressed in frog oocytes following, for example, injection of Xenopus neuroD1 RNA into one of the two cells in a two-cell stage Xenopus embryo, and monitoring expression of neuronal-specific genes in the injected as compared to uninjected side of the embryo by immunochemistry or in situ hybridization.
xe2x80x9cOver-expressionxe2x80x9d means an increased level of a neuroD protein or of neuroD transcripts in a recombinant transformed host cell or in a tumor cell relative to the level of protein or transcripts in the untransformed host cell or in the normal cell from which the tumor is derived.
As noted above, the present invention provides isolated and purified polynucleotide molecules encoding various members of the neuroD family. The disclosed sequences may be used to identify and isolate additional neuroD polynucleotide molecules from suitable mammalian or non-mammalian host cells such as canine, ovine, bovine, caprine, lagomorph, or avian. In particular, the nucleotide sequences encoding the HLH region may be used to identify polynucleotide molecules encoding other proteins of the neuroD family. Complementary DNA molecules encoding neuroD family members may be obtained by constructing a cDNA library mRNA from, for example, fetal brain, newborn brain, and adult brain tissues. DNA molecules encoding neuroD family members may be isolated from such a library using the disclosed sequences to provide probes to be used in standard hybridization methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference), and Bothwell, Yancopoulos and Alt, ibid.) or by amplification of sequences using polymerase chain reaction (PCR) amplification (e.g., Loh et al., Science 243:217-222, 1989; Frohman et al., Proc. Natl. Acad Sci. USA 85:8998-9002, 1988; Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, 1989; and Mullis et al., PCR: The Polymerase Chain Reaction, 1994, which are incorporated by reference herein in their entirety). In a similar manner, genomic DNA encoding neuroD proteins may be obtained using probes designed from the sequences disclosed herein. Suitable probes for use in identifying neuroD genes or transcripts may be obtained from neuroD-specific sequences that are highly conserved regions between mammalian and amphibian neuroD coding sequences. Nucleotide sequences, for example, from the region encoding the approximately 40 residues following the helix-2 domain are suitable for use in designing PCR primers. Alternatively, oligonucleotides containing specific DNA sequences from a human neuroD1, neuroD2, or neuroD3 coding region may be used within the described methods to identify related human neuroD genomic and cDNA clones. Upstream regulatory regions of neuroD may be obtained using the same methods. Suitable PCR primers are between 7-50 nucleotides in length, more preferably between 15 and 25 nucleotides in length. Typically, probes must be at least 10 nucleotides in length to form stable hybrids. Alternatively, neuroD polynucleotide molecules may be isolated using standard hybridization techniques with probes of at least about 15 nucleotides in length and up to and including the full coding sequence. Southern analysis of mouse genomic DNA probed with the murine neuroD1 cDNA under stringent conditions showed the presence of only one gene, suggesting that under stringent conditions bHLH genes from other protein families will not be identified. Other members of the neuroD family can be identified using degenerate oligonucleotides based on the sequences disclosed herein for PCR amplification or by hybridization at moderate stringency using probes based on the disclosed sequences.
The regulatory regions of neuroD may be useful as tissue-specific promoters. Such regulatory regions may find use in, for example, gene therapy to drive the tissue-specific expression of heterologous genes in pancreatic, gastrointestinal, or neural cells, tissues or cell lines. As shown in Example 14, murine neuroD1 promoter sequences reside within the 1.4 kb 5xe2x80x2 untranslated region. Regulatory sequences within this region are identified by comparison to other promoter sequences and/or deletion analysis of the region itself.
In other aspects of the invention, a DNA molecule coding a neuroD protein is inserted into a suitable expression vector, which is in turn used to transfect or transform a suitable host cell. Suitable expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a polynucleotide molecule of interest in a host cell and may also include a transcription termination signal, these elements being operably linked in the vector. Representative expression vectors may include both plasmid and/or viral vector sequences. Suitable vectors include retroviral vectors, vaccinia viral vectors, CMV viral vectors, BLUESCRIPT vectors, baculovirus vectors, and the like. Promoters capable of directing the transcription of a cloned gene or cDNA may be inducible or constitutive promoters and include viral and cellular promoters. For expression in mammalian host cells, suitable viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981). Suitable cellular promoters for expression of proteins in mammalian host cells include the mouse metallothionine-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), a mouse Vk promoter (13ergman et al., Proc. Natl. Acad Sci. USA 81:7041-7045, 1983; Grant et al. Nucleic Acid Res. 15:5496, 1987), and tetracycline-responsive promoter (Gossen and Bujard, Proc. Natl. Acad Sci. USA 89:5547-5551, 1992, and Pescini et al., Biochem. Biophys. Res. Comm. 202:1664-1667, 1994). Also contained in the expression vectors, typically, is a transcription termination signal located downstream of the coding sequence of interest. Suitable transcription termination signals include the early or late polyadenylation signals from SV40 (Kaufinan and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982), the polyadenylation signal from the Adenovirus 5 e1B region, and the human growth hormone gene terminator (DeNoto et al., Nucleic Acid Res. 9:3719-3730, 1981). Mammalian cells, for example, may be transfected by a number of methods including calcium phosphate precipitation (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), lipofection, microinjection, and electroporation (Neumann et al., EMBO J. 1:8410845, 1982). Mammalian cells can be transduced with viruses such as SV40, CMV, and the like. In the case of viral vectors, cloned DNA molecules may be introduced by infection of susceptible cells with viral particles. Retroviral vectors may be preferred for use in expressing neuroD proteins in mammalian cells particularly if the neuroD genes used for gene therapy (for review, see, Miller et al. Methods in Enzymology 217:581-599, 1994; which is incorporated herein by reference in its entirety). It may be preferable to use a selectable marker to identify cells that contain the cloned DNA. Selectable markers are generally introduced into the cells along with the cloned DNA molecules and include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. Selectable markers may also complement auxotrophs in the host cell. Yet other selectable markers provide detectable signals, such as xcex2-galactosidase to identify cells containing the cloned DNA molecules. Selectable markers may be amplifiable. Such amplifiable selectable markers may be used to amplify the number of sequences integrated into the host genome.
As would be evident to one of ordinary skill in the art, the polynucleotide molecules of the present invention may be expressed in Saccharomyces cerevisiae, filamentous fungi, and E. coli. Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (see, xe2x80x9cGene Expression Technology,xe2x80x9d Methods in Enzymology, Vol. 185, Goeddel (ed.), Academic Press, San Diego, Calif., 1990; and xe2x80x9cGuide to Yeast Genetics,and Molecular Biology,xe2x80x9d Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San Diego, Calif., 1991, which are incorporated herein by reference). Filamentous fungi may also be used to express the proteins of the present invention; for example, strains of the fungi Aspergillus (McKnight et al., U.S. Pat. No.4,935,349, which is incorporated herein by reference). Methods for expressing genes and cDNAs in cultured mammalian cells and in E. coli are discussed in detail in Sambrook et al., 1989. As will be evident to one skilled in the art, one can express the protein of the insant invention in other host cells such as avian, insect, and plant cells using regulatory sequences, vectors and methods well established in the literature.
NeuroD proteins produced according to the present invention may be purified using a number of established methods such as affinity chromatography using anti-neuroD antibodies coupled to a solid support. Fusion proteins of antigenic tag and neuroD can be purified using antibodies to the tag. Additional purification may be achieved using conventional purification means such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982, which is incorporated herein by reference) and may be applied to the purification of recombinant neuroD described herein.
The invention provides isolated and purified polynucleotide molecules encoding neuroD proteins that are capable of hybridizing under stringent conditions to an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and/or SEQ ID NO:16, and also including the polynucleotide molecules complementary to the coding strands. The subject isolated neuroD polynucleotide molecules preferably encode neuroD proteins that trigger differentiation in ectodermal cells, particularly neuroectodermal stem cells, and in more committed cells of that lineage, for example, epidermal precursor cells, pancreatic and gastrointestinal cells. Such neuroD expression products typically form heterodimeric bHLH protein complexes that bind in the 5xe2x80x2-regulatory regions of target genes and enhance or suppress transcription of the target gene.
In some instances, cancer cells may contain a non-functional neuroD protein or may contain no neuroD protein due to genetic mutation or somatic mutations such that these cells fail to differentiate. For cancers of this type, the cancer cells may be treated in a manner to cause the over-expression of wild-type neuroD protein to force differentiation of the cancer cells. Detection of overexpressed neuroD or other neurogenic differentiation factors may serve to identify different types of brain tumors.
Antisense neuroD nucleotide sequences, that is, nucleotide sequences complementary to the non-transcribed strand of a neuroD gene, may be used to block expression of mutant neuroD expression in neuronal precursor cells to generate and harvest neuronal stem cells or, alternatively, to suppress inappropriately expressed neuroD in tumor cells. The use of antisense oligonucleotides and their applications have been reviewed in the literature (see, for example, Mol and Van der Krul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, N.Y., 1992; which is incorporated by reference herein in its entirety). Suitable antisense oligonucleotides are at least 11 nucleotide in length and may include untranslated (upstream or intron) and associated coding sequences. Suitable target sequences for antisense oligonucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, sites that interfere with ribosome progression, and 5xe2x80x2 untranslated regions (promoter/enhancer) of the target gene. Antisense oligonucleotides may be prepared synthetically or by the insertion of a DNA molecule containing the target DNA sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself The expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense oligonucleotides. Synthetic oligonucleotides may be introduced, e.g., by electroporation, calcium phosphate precipitation, liposomes, or microinjection. Synthetic antisense oligonucleotides may be stabilized, e.g., by using intercalating agents that are covalently attached to either or both ends of the oligonucleotide, or by being made nuclease resistant by modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates, phosphorodithioates, or by using alpha-anomers of the deoxyribonucleotides.
NeuroD proteins bind to 5xe2x80x2 regulatory regions of neurogenic genes that are involved in neuroectodermal differentiation, including development of neural and endocrine tissues. As described in the Examples given below, murine neuroD1 has been detected in neuronal, pancreatic and gastrointestinal tissues in embryonic and adult mice suggesting that neuroD1 functions in the transcription regulation in these tissues. NeuroD proteins alter the expression of subject genes by, for example, down-regulating or up-regulating transcription, or by inducing a change in transcription to an alternative open reading frame.
DNA sequences recognized by the various neuroD proteins may be determined using a number of methods known in the literature including immunoprecipitation (Biedenkapp et al., Nature 335:835-837, 1988; Kinzler and Vorgelstein, Nuc. Acids Res. 17:3645-3653, 1989; and Sompayrac and Danna, Proc. Natl. Acad Sci. USA 87:3274-3278, 1990; which are incorporated by reference herein), protein affinity columns (Oliphant et al., Mol. Cell Biol. 9:2944-2949, 1989; which is incorporated by reference herein), gel mobility shifts (Blackwell and Weintraub, Science 250:1104-1110, 1990; which is incorporated by reference herein), and Southwestern blots (Keller and Maniatis, Nuc. Acids Res. 17:4675-4680, 1991; which is incorporated by reference herein).
One embodiment of the present invention involves the construction of inter-species hybrid neuroD proteins and hybrid neuroD proteins containing at least one domain from two or more neuroD family members to facilitate structure-function analyses or to alter neuroD activity by increasing or decreasing the neuroD-mediated transcriptional activation of neurogenic genes relative to the wild-type neuroD(s). Hybrid proteins of the present invention may contain the replacement of one or more contiguous amino acids of the native neuroD protein with the analogous amino acid(s) of neuroD from another species or other protein of the neuroD family. Such interspecies or interfamily hybrid proteins include hybrids having whole or partial domain replacements. Such hybrid proteins are obtained using recombinant DNA techniques. Briefly, DNA molecules encoding the hybrid neuroD proteins of interest are prepared using generally available methods such as PCR mutagenesis, site-directed mutagenesis, and/or restriction digestion and ligation. The hybrid DNA is then inserted into expression vectors and introduced into suitable host cells. The biological activity may be assessed essentially as described in the assays set forth in more detail in the Examples that follow.
The invention also provides synthetic peptides, recombinantly derived peptides, fusion proteins, and the like that include a portion of neuroD or the entire protein. The subject peptides have an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions with an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. Representative amino acid sequences of the subject peptides are disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO: 17. The subject peptides find a variety of uses, including preparation of specific antibodies and preparation of agonists and antagonists of neuroD activity.
As noted above, the invention provides antibodies that bind to neuroD proteins. The production of non-human antisera or monoclonal antibodies (e.g., murine, lagormorph, porcine, equine) is well known and may be accomplished by, for example, immunizing an animal with neuroD protein or peptides. For the production of monoclonal antibodies, antibody producing cells are obtained from immunized animals, immortalized and screened, or screened first for the production of the antibody that binds to the neuroD protein or peptides and then immortalized. It may be desirable to transfer the antigen binding regions (e.g., F(abxe2x80x2)2 or hypervariable regions) of non-human antibodies into the framework of a human antibody by recombinant DNA techniques to produce a substantially human molecule. Methods for producing such xe2x80x9chumanizedxe2x80x9d molecules are generally well known and described in, for example, U.S. Pat. No. 4,816,397; which is incorporated by reference herein in its entirety. Alternatively, a human monoclonal antibody or portions thereof may be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to the neuroD family member, e.g., according to the method generally set forth by Huse et al. (Science 246:1275-1281, 1989, which is incorporated by reference herein in its entirety). The DNA molecule may then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity.
The invention also provides methods for inducing the expression of genes, such as neurotransmitters or neuromodulatory factors, that are associated with neuronal phenotype in a cell that does not normally express those genes. For example, the modulation of gene expression by neuroD can be carried out in cells of the neuroectodermal lineage, glial cells, neural crest cells, and epidermal epithelial basal stem cells, and all types of both mesodermal and endodermal lineage cells. NeuroD expression may also be used as a means of inducing expression of genes associated with pancreatic and gastrointestinal phenotype, e.g., insulin or gastrointestinal-specific enzymes.
As illustrated in Example 10, the expression of Xenopus neuroD1 protein in stem cells causes redirection of epidermal cell differentiation and induces terminal differentiation into neurons, i.e., instead of epidermal cells. Epithelial basal stem cells (i.e., in skin and mucosal tissues) are one of the few continuously regenerating cell types in an adult mammal. Introduction of the subject nucleotide sequences into an epithelial basal stem cell may be designed to achieve transient, constitutive, or regulated expression, and may be accomplished in vitro or in vivo using a suitable gene therapy vector delivery system (e.g., a retroviral vector), a microinjection technique (see, for example, Tam, Basic Life Sciences 37:187-194, 1986, which is incorporated by reference herein in its entirety), or a transfection method (e.g., naked or liposome encapsulated DNA or RNA; see, for example, Trends in Genetics 5:138, 1989; Chen and Okayama, Biotechniques 6:632-638, 1988; Mannino and Gould-Fogerite, Biotechniques 6:682-690, 1988; Kojima et al., Biochem. Biophys. Res. Comm. 207:8-12, 1995; which are incorporated by reference herein in their entirety).
Transformed host cells of the present invention are useful in vitro as convenient sources of neuronal and other growth factors for screening anti-cancer drugs capable of driving terminal differentiation in neural tumors, as sources of recombinantly expressed neuroD protein for use as an antigen in preparing monoclonal and polyclonal antibodies useful in diagnostic assays, and for screening for compounds capable of increasing or decreasing the activity of neuroD.
Transformed host cells of the present invention are also useful in vivo for transplantation at sites of traumatic neural injury where motor or sensory neural activity has been lost, e.g., for treating patients with hearing or vision loss due to optical or auditory nerve damage, patients with peripheral nerve damage and loss of motor or sensory neural activity, and patients with brain or spinal cord damage from traumatic injury or radiation injury. For example, donor cells from a patient such as epithelial basal stem cells are cultured in vitro and then transformed or transduced with a neuroD nucleotide sequence. The transformed cells are then returned to the patient by microinjection at the site of neural dysfunction. In addition, as neuroD appears capable of regulating expression of insulin, transformed host cells of the present invention may be useful for transplantation into patients with diabetes. For example, donor cells from a patient such as fibroblasts, pancreatic islet cells, or other pancreatic cells are harvested and transformed or transfected with a neuroD nucleotide sequence. The genetically engineered cells are then returned to the patient. In another embodiment, such engineered host cells may find use in the treatment of malabsorption syndromes or gastrointestinal dysmotility syndromes (Hirsh Prung""s Disease).
Furthermore, the nucleotide sequences of the subject invention are useful for constructing cDNA and oligonucleotide probes for Northern or Southern blots, dot-blots, or PCR assays for identifying and quantifying the level of expression of neuroD in a cell. In addition, birth defects and spontaneous abortions may result from expression of an abnormal neuroD protein; thus, screening neuroD expression may be useful in prenatal screening of mothers in utero.
The neuroD sequences of the subject invention are also useful for constructing recombinant cell lines, ova, and transgenic embryos and animals including dominant-negative and xe2x80x9cknock-outxe2x80x9d recombinant cell lines in which the transcription regulatory activity of neuroD protein is down-regulated or eliminated. Such cells may contain altered neuroD coding sequences that result in the expression of a defective neuroD protein that is not capable of enhancing, suppressing or activating transcription of its target gene(s). The subject cell lines and animals find uses in screening for candidate therapeutic agents capable of either substituting for neuroD or correcting the cellular defect caused by a defective neuroD. Alternatively, cell lines expressing wild-type neuroD proteins may be useful for correcting birth defects that result from defective neuroD expression.
In addition, neuroD polynucleotide molecules may be joined to reporter genes, such as xcex2-galactosidase or luciferase, and inserted into the genome of a suitable embryonic host cell such as a mouse embryonic stem cell by, for example, homologous recombination (for review, see Capecchi, Trends in Genetics 5:70-76, 1989; which is incorporated by reference). Cells expressing neuroD may then be obtained by subjecting the differentiating embryonic cells to cell sorting, leading to the purification of a population neuroblasts that are useful for studying neuroblast sensitivity to growth factors or chemotherapeutic agents that may be used as a source of neuroblast-specific protein products or gene transcripts.
As illustrated in Example 14, neuroD1 xe2x80x9cknock-outxe2x80x9d mice had diabetes, as demonstrated by blood glucose levels 2 and 3 times that of wild-type mice, and they died within four days of birth, while heterozygous mutants exhibited wild-type blood glucose levels. These results suggest that neuroD1 xe2x80x9cknock-outxe2x80x9d mice may be useful for studying methods to rescue homozygous mutants and as hosts to test transplant tissue for treating diabetes. These results suggest further that in vivo correction of neuroD 1 deficiencies may therapeutically benefit diabetes patients.
The subject neuroD genes also are useful for constructing gene transfer vectors (e.g., retroviral vectors, and the like) wherein neuroD is inserted into the coding region of the vector under the control of a promoter. NeuroD gene therapy may be used to correct traumatic neural injury that has resulted in loss of motor or sensory neural function. For these therapies, gene transfer vectors may either be injected directly at the site of the traumatic injury, or the vectors may be used to construct transformed host cells that are then injected at the site of the traumatic injury. The results disclosed in Example 10 indicate that introduction of neuroD1 induces a non-neuronal cell to become a neuron, suggesting that transplantation and/or gene therapy with neuroD1 could be used to repair neural defects resulting from traumatic injury. NeuroD1 also may be useful for treating neurological disorders such as Alzheimer""s disease, Huntington""s disease, and Parkinson""s disease, in which a population of neurons have been damaged. For such therapies, recombinant neuroD1 sequences may be introduced into existing neurons, or endogenous neuroD1 expression is induced in existing neurons in vivo. Alternatively, neuroD1 expression is induced in non-neuronal cells (e.g., glial cells in the brain or basal epithelial cells) to induce expression of genes that confer a complete or partial neuronal phenotype that ameliorates aspects of the disease. As an example, Parkinson""s disease is caused, at least in part, by the death of neurons that supply the neurotransmitter dopamine to the basal ganglia. Increasing the levels of neurotransmitter ameliorates the symptoms of Parkinson""s disease. Expression of neuroD1 in basal ganglia neurons or glial cells may induce aspects of a neuronal phenotype such that the neurotransmitter dopamine is produced directly in these cells. Alternatively, donor cells expressing a neuroD gene could be transplanted into the affected region. Also, neuroD1 can be expressed in non-pancreatic cells to induce expression of genes that confer a complete or partial pancreatic phenotype that ameliorates aspects of diabetes. Within yet another embodiment, neuroD1 is expressed in pancreatic islet cells to induce expression of genes that induce the expression of insulin.
The subject neuroD genes also are useful for the preparation of transplantable recombinant neuronal precursor cell populations from embryonic ectodermal cells, non-neural basal stem cells, and the like. The isolated polynucleotide molecules encoding neuroD proteins of the present invention permit the establishment of primary (or continuous) cultures of proliferating embryonic neuronal stem cells under conditions mimicking those that are active in development and cancer. The resultant cell lines find uses: i) as sources of novel neural growth factors, ii) in screening assays for anti-cancer compounds, and iii) in assays for identifying novel neuronal growth factors. For example, a high level of expression of neuroD was observed in the embryonic optic tectum, indicating that neuroD1 protein may regulate expression of factors trophic for growing retinal cells. Such cells may be useful sources of growth factors, and may be useful in screening assays for candidate therapeutic compounds.
The cell lines and transcription regulatory factors disclosed herein offer the unique advantage that since they are active very early in embryonic differentiation they represent potential switches, e.g., ONxe2x86x92OFF or OFFxe2x86x92ON, controlling subsequent cell fate. If the switch can be shown to be reversible (i.e., ON⇄OFF), the neuroD genes and proteins disclosed herein provide exciting opportunities for restoring lost neural and/or endocrine functions in a subject.